Various scientific and scholarly articles are referred to throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
Plants defend themselves against pathogens through both pre-formed and inducible resistance mechanisms. Among the inducible responses are the hypersensitive resistance (HR) response and systemic acquired resistance (SAR). The HR is a localized plant response characterized by a suite of physiological changes culminating in plant cell death and cessation of pathogen growth. SAR is a systemic resistance response that is induced after formation of a necrotic lesion, either as part of the HR or as a symptom of disease. Although the HR and SAR have been the major forms of induced plant resistance studied, evidence for other resistance mechanisms exists.
The HR can be induced by the interaction between a plant resistance gene and a matching pathogen avirulence gene. Such “gene-for-gene” interactions provide a narrow range of resistance as they differentiate between races of a pathogen based on expression of a specific avirulence gene. Resistance gene products are thought to function as receptors for ligands produced directly or indirectly by avirulence genes. Multiple biochemical events are associated with the HR, including an oxidative burst, K/Cl ion exchange, deposition of autofluorescent compounds and callose in the cell wall, synthesis of antimicrobial phytoalexins, and cell death.
In Arabidopsis, SAR is associated with the expression of three pathogenesis-related genes: PR-1 (unknown function), BGL2 (β-glucanase, also known as PR-2) and PR-5 (a thaumatin-like protein). Arabidopsis mutants identified based upon constitutive expression of PR genes (cpr1 and cpr5) are resistant to the fungal pathogen Peronospora parasitica and the bacterial pathogen Pseudomonas syringae pv maculicola. Other mutants that constitutively express PR genes have been isolated based upon the development of spontaneous leaf lesions that are similar in appearance to the lesions of an HR. These lesion mimic mutants also show resistance to both fungal and bacterial pathogens.
Methyl jasmonate and ethylene may induce a defense pathway that is independent of SA. Wounding as well as pathogen attack induce the production of jasmonic acid, which in turn induces defense genes other than those associated with SAR, including genes that encode defensins and thionins. Defensins and thionins are low molecular weight polypeptides that have potent antimicrobial activity in vitro. Arabidopsis plants over-expressing endogenous thionin have increased resistance to the fungal pathogen Fusarium oxysporum. Transgenic Arabidopsis plants unable to accumulate SA and thus unable to express SAR are able to respond to the jasmonic acid signal and express both defensin and thionin genes. Mutants that constitutively express the proposed jasmonic acid pathway, but not the SA pathway have not been reported; however, the cpr5 and acd2 mutants of Arabidopsis constitutively expresses both PR genes and defensin.
Another defense pathway that is independent of SA is induced by the biocontrol bacterium P. fluorescens and is termed induced systemic resistance (ISR) (Pieterse, et al., 1996, Plant Cell 8:1225–1237). ISR is observed when Arabidopsis plants grown in soil containing P. fluorescens are challenged with virulent bacterial and fungal pathogens. Under these conditions, the Arabidopsis plants develop less severe disease symptoms than do control plants grown in soil alone. ISR is not associated with the expression of PR genes and is observed in plants unable to accumulate SA, indicating that this pathway is independent of SAR (Pieterse, et al., 1996, supra). It has not been determined whether the proposed jasmonic acid pathway contributes to ISR.
Screens for plant mutants that display enhanced resistance to virulent pathogens have been performed with several crop species. From these studies, barley resistant to powdery mildew (the mlo mutation), sugar-cane resistant to smut, mulberry resistant or tolerant to nematodes, mulberry resistant to Dogare disease and peppermint resistant to Verticillium wilt were identified. Of these, only the mlo resistance has been well characterized.
The mlo mutation of barley mediates resistance to all common races of the powdery mildew fungus Erysiphe graminis f sp hordei and, thus, provides a broader spectrum resistance than do the gene-for-gene type of resistance genes (Jorgensen, 1992, Euphytica 63:141–152).
Resistance in mlo mutants correlates with the formation of cell wall appositions that may prevent fungal penetration (Jorgensen, 1992, supra; Wolter et al., 1993, Mol. Gen. Genet. 239:122–128) and with plant cell death (Peterhänsel et al., 1997, Plant Cell 9:1397–1409). Defense genes are not constitutively expressed in mlo mutant barley; however, they are induced more rapidly upon infection by E. graminis (Peterhänsel, et al., 1997, supra). The wild-type Mlo gene has been cloned and is hypothesized to be a negative regulator of defense responses such that mutant mlo alleles mediate resistance by allowing abnormal defense responses to occur both spontaneously and during an E. g. hordei infection (Wolter, et al., 1993, supra; Buschges et al., 1997, Cell 88:695–705).
The isolation of novel mutants and genes that control defense responses will broaden the range of affected pathogens. It would be particularly advantageous to isolate mutants or genes involved in inducibly enhanced disease resistance without spontaneously occurring abnormal defense responses. Novel regulatory mutants are likely to have distinct pathogen ranges due to differential induction of unique subsets of genes. The different enhanced response may yield resistance responses that are spatially distinct (i.e. epidermal v. mesophyll cells), temporally distinct (i.e. affecting early v. late stages of infection), and/or comprised of a distinct subset of defense mechanisms (i.e. formation of necrotic lesions, deposition of callose, synthesis of antimicrobial Phytoalexins and others). The isolation of mutants will yield the critical gene(s), which can be used to transgenically transfer the enhanced resistance trait to new species.